Netropsin and process for its production



Feb. 19, 1952 Filed Aug. 25, 1949 FIG. 1 INFRARED ABSORPTION SPECTRUM 0F NETROPSIN HYDROCHLORIDE saaesae-sa A. c. FINLAY ETAL 2,586,762

NETROPSIN AND PROCESS FOR ITS PRODUCTION 2 SHEETSSHEET l 0 E I350 I300 I250 I200 H50 H00 mso moo s50 son 850 B00 150 100 650 FREQUENCY IN, CM'.

PERCENT TRANSMITTANCE IN V EN TORS m CUM.-

T/{f/f? ATTORNEY Feb. 19, 1952 Filed Aug. 25, 1949 F] G 2. INFRARED ABSORPTION SPECTRUM 0F NETROPSIN SULFATE A. C. FlNLAY ETAL NETROPSIN AND PROCESS FOR ITS PRODUCTION 2 SHEETS-SHEET 2 I250 I200 H50 "00 I050 I000 950 900 850 800 FREQUENCY IN CM PERCENT TRANSMITTANCE v IN l E'/|/TO,?$-- ALEXANDER C.FINLAY 8| BEN A. SOBIN BY C THE/R ATTORNEY Patented Feb. 19, 1952 NETROPSIN AND PROCESS FOR ITS PRODUCTION Alexander 0. Finlay, Long Island City, and Ben A. Sobin, New York, N. Y., assignors to Chas. Pfizer & 00., Inc., Brooklyn, N. Y., a corporation of Delaware Application August 25, 1949, Serial No. 112,411

8 Claims. (01. 167-65) This invention is concerned with a new and useful antibiotic substance, Netropsin, and a process for preparing the same by the cultivation under particular controlled conditions of certain strains of a micro-organism hitherto undescribed, called Streptomyces netropsis. This organism was isolated from a sample of soil taken near Hudson, New York, and its description, following the key of Bergeys Manual of Determinative Bacteriology, sixth edition, page 929-933 follows:

The cultural characteristics of this new species, based on our isolate .number 2937-6, are given below in tabular .form. .A living culture of said isolate ,has been deposited with the Fermentation Division of the Northern Regional Research Laboratory at Peoria, Illinois, and has been added to its-permanentcollection of microorganisms as NRRL-2268. The colorsywhere R. is written, are those of Ridgway, Color Standards and Nomenclature.

Color Amount-of Medium Gmwth Aerial Mycelium Soluble and Spores Pigment Glucose aspara- Moderate".-. White aerial my- Brown.

gme celium; no

spores.

Other liemarks. -Oolonies slightly elevated; edge smooth; surface rough, slightly wrmkled; reverse brown; whorls or terminal clusters of short, stiff hyphae n tips of short hyphae (synthetic agar). Oonid- 1a borne in chains, gram-variable, approximately 0.65 x 1.30;, short cylind rical (conidial data from synthetic agar tubes) Colonies plated out all were similar; most of colony was White with some Light Mouse Gray and Cream Bufi (R); reverse from Ivory Yellow to Honey Yellow (R).

Gelatin Moderate White aerial my- D a r l:

celium. brown.

Other Remarks-No liquefaction. Reverse brown.

Litmus milk Very poor None.

Other Remarks.-pH-unchanged. N0 ,peptonization or hydrolysis.

Ca malate Poor to mod- White aerial my- None.

erate. celium.

Other Remarks-Reverse Cream Bufl (R).

Glucose agar Good White aerial my- Brown.

celium.

Other Remarks-Reverse dark brown.

Potato Poor Bully-Brown (R); .Mediumto no aerial. myceld .a r k ium, waxy, brown.

I wrinkled.

Starch plates. "n- M o d e rate White aerial vmy- None.

thin. celium'.

Other Remarks.Reverse Pale Olive Buff (R). Starch strongly hydrolyzed.

Color Amount of 1 Medium Growth Aerial Mycelium Soluble and Spores .Plgment Synthetic agar. Poor, thin' Pale Vinaceous None.

' Fawn (Risporulation.

OthcrRemarks-Reverse Pale Olive Buff (R).

Nutrient agar"... Moderate to White'aerial my- L i g h t good. celium. brown.

Other Remarks-Reverse light brown.

Emersons Good A. White aerial my- L i g h t celium. brown.

Other Remarks-Reverse light brown.

Nitrate broth"-.. Good Brown.

Other Remarks-Nitrates not reduced.

It is tobe understood that for the production of Netropsin we are not limited to this particular organism or organisms obtained from Antibiotics produced by organisms of a genus Streptomyces of the Actinomycetes of which a number are now well known fall into two classes, (1) neutral substances extractable from broths at acid, neutral and alkaline pH by solvents and" (2) basic substances in general not extractable by organic solvents. Chloromycetin and Actinomycin are typical of the first group and Streptomycin and Streptothricin of the second. By this criterion, Netropsin is a member of the second group. Netropsin also shares with this group the property of being precipitated by certain acid dyestuffs such as, Erio Chrome Violet, Naphthol Blue Black, Orange II and other similar sulfonic acid dyes. This second group is characterized by a wide antibiotic spectrum, particularly among the Gram-negative bacteria. The following table shows the comparative spectra of Streptomycin, Streptothricin, Chloromyc'etin, Aureomycin and Netropsin.

TABLE I Mcy. per ml. of antibiotic necessary to inhibit growth of micro-organisms on nutrient agar plates Organism Aureo- Chloro- Strepto- Strepto- Netropmycin 1 mycetin 1 mycin a thricin 4 sin I S. aureus 0. 9 7 4 6 5 0.9 7 6 5 3 1 5 5 30 5 0.9 5 7 70 20 l 500 1, 000 10 30 5 4 10 10 10 2 5 30 5 8 5 5 20 5 20 5 5 30 10 10 7 5 10 5 l 1 7 7 5 5 5 7 7 A. aer0gencs 0. 9 2 5 5 5 Pa. aeruainos 10 10 10 1, 000 Proteus sp 50 80 10 8 40 M. albicana 2, 000 2,000 2, 000 100 90 1 Crystalline hydrochloride.

, I Crystalline Streptomycin sulfate (750 streptomycin units/mg.) expressed as meg. active base.

l Streptothriciu sulfate (excrystalline hellanthate; 809'streptornycin units/ 1115.).

I Crystalline N etropsin hydrochloride.

Another method of distinguishing Netropsin from known-antibiotics produced by species of Streptomyces is by the action of antibiotics on strains of bacteria made resistant to the various antibiotics.

In all cases the dosage has been reduced to E. coli dilution units per milligram. By E. coli dilution units (CDU) per milligram, we mean Netropsin is advisably isolated in the form of its salts, preferably the sulfate or the hydrochloride. An examination of Netropsin hydrochloride indicates the following characteristics. Upon drying at 100 C. for 5 hours there is a loss in weight of about 7.8% and loss of crystallinity without loss in antibacterial activity. The dried material gave the following elementary analysis.

the volume of nutrient broth in milliliters to C Per cent 429 which one milligram of the antibiotic prepara- H do tion (which may be of varying degrees of purity) N 1 may be diluted, when inoculated'with a 10- C1 dilution of an 18 hour culture of E. coli grown 01 (total) under the same conditions and at the end of 18 hours incubation at 37 C., shows no growth. In Table 11 it may be seen that Netropsin is able to restrain the growth of strains of A. aerogenes made resistant to Streptomycin, Streptothricin, and Chloromycetin. This clearly distinguishes Netropsin from these antibiotics.

TABLE II [a]n=0(1% in H2O, 23 C.)

Comparison of action of various antibiotics against strains of A. aerogenes in nutrient broth Aureomycin Streptomycin Streptothri- Chloromyoe- Netropsin Organism 00 50 100 cin 50 100 tin 50 100 50 100 (CDU/ml.) (CDU/ml.) (CDU/ml.) (CDU/ml.) (CDU/ml.)

A. aeronenes strain 'A A. aeroaenes strain B A. aeroaenes strain 0 A. aerogenes strain D g A. aerogenes strain A is sensitive to 25 ODU/ml. of all antibiotics. A. aerogenes strain B is resistant to 200 ODU/ml. of chloromycetin.

A. aerogenes strain 0 is resistant to 4400 CDU/ml. of streptomycin and ca. 25 CDU/ml. of streptothricin.

tomycin.

The toxicity of various antibiotics including formamide solutions. The ultraviolet absorp- Netropsin is given in Table III.

TABLE III Toxicity of various antibiotics (Mg. per 20 g. Mouse] V I tion spectrum of an aqueous solution shows the following maxima:

li... 8 ma=430 A mineral oil mull of the anhydrous Netropsin hydrochloride shows many characteristic absorption maxima in the infrared region. Among these are the following frequencies (in reciprocal centimeters): 3525, 3300, 3170, 1703, 1e70, 1583,

1518, 1300, 1244,1211, 1149, 1104, 1068, 1069, 1.031, 1021, 993, 967, 898,852, 835,818, 798, 778, 751, 734, 679. The infrared absorption spectrum of this mineral oil mull within the characteristic 7e region of wave numbers between 1350 and 650 em." is .shown in Fig.1 of the. accompanying drawings.

Before. drying the crystals of Netropsin hydrochloride are long, thin, colorless, hydrated prisms which exhibit oblique extinction- The stability of Netropsin hydrochloride in water .at .500 meg/ml. when boiled for .flfteen minutes is; as vfollows:

1 .Per Cent pH Loss in Activity When Netropsin. hydrochloride is dissolved in an equivalent quantity of ,0.2*I-Iv sodium hydroxide,

inactivation .is complete in less thantwo hours.

Netrops in sulfate is somewhat more satisfactel-yfrom a manipulative standpoint than the hydrochloride. .It may be prepared by the addition. of 0Z65. g. of potassium sulfate. in 10 ml. of water to 1.0g. of Netropsin hydrochloride in '75 ml. of boiling water. on cooling, the hydrated sulfate is recovered aslong, colorless needles in a virtually quantitative yield. A recrystallized sample. driedat 100 C. and 0-.02 mm. Hg ,presit... lit-2st m,.=429 li... at 2aemt=ise The; infraredzabsorption spectrum. of. a. mineral oil mull of. hydrated Netropsin sulfate exhibits the, broad .maxima characteristic of large. molecules at approximately the following wave;

lengths: 329.0,.1580. 126.0,. 11.455, 1.100,. 1050,1990, 960, 89.0, 850., 830 and 805. cmr In addition,

there are; certain,somewhat-sharper peaks at approximately the following wave lengths: 1660,. 16.30;-1400, 128.5. and1l21.0-cm.- Thecharacter- I istic: portion. of: thisriniirared absorption. spectrum.

within. the range of. from: 1350 to 650. charshown in ,Fig.. 2. of the drawings.

The ireehase: oiNetropsin'is diificult. to isolate owingrtoiits; instability; The analyses .andrmolecular weights of itssalts .indicateitto be altetra acidic base, probably corresponding, to the ;:ormulazr CaIEIAaNiaOA. Itisrelatively stable in-acid solution, having a half. life of about two. hours in: 1. N sulfuric acid-at: 100 C.

The behavior of severalantibiotics on .apaper chromatogram in a system. of water saturated with n-butanol containing 2.1% p-toluenesulfonic acid run'for 24. hours at 23 C was. compared using, B. subtilz's as. the test organism,

Antibiotic. Rf

Streptomycin AM..." 0:24 Streptothr1cin 0. 025 Netropsiu 0.23

Netropsin and its salts have not yet been dem-- onstrated to be useful in human. therapy. However,:it.has been. demonstrated thatcthc Netropsin hydrochloride is highlyeflective for protecting wool fabrics against attack by clothes moth larvae.

This invention embraces the .processior igrowinga new and hithertountiescribedmicrmorganismrat 2.440 C. under. submerged-conditions of agitation and :aeration :onimedia consisting of. a carbohydrate source, :such as. sugars, starch, glycerol; an orgamcinitrogemsource,'such assaybean; meal orwheat gluten: a sourceofgmwth substances, such as distillei's solublesa; common salt and calcium carbonate as :abuifering "agent, separating the mycelium after growth has been completed, precipitating the antibioticout of the brothubyzthe addition of su-lfonic acid dyestuffs, such as. Erio Chrome Violet, Naphthol: Blue Black, Orange II at a specifiedpI-I, decomposing the dye cake and removinggthe antibiotic 'from the filtrate. The inventionf is' not. l mited, however, to any specific method antibiotic: recovery.. The new antibiotic, Netrnpsin ,v produced as afore said, possesses unique. and? valuable properties which difierentiate itfromall knownrandpreviously described antibiotics.

Inoculum is obtainedaby employing a growth from slants or Roux bottles inoculated with Streptomyces netropsis, A solid. medium. suitable for this initial growth G.. Dextrose ..r- .l .10

Beef extract 4 Peptone 4 Yeast extract l Sodium chloride 2.5

Distilled water to 1000. ml.; adjust'to 7.0- and add- Agar 30 will usually be at the most favorable period in two days. From the inoculum tank the broth mixed with the micro-organism is forced into the fermenter under completely sterile conditions: and growth is continued for-a "'rtherperiod-of 2 days. At all times aeration is maintained in tanks by blowingin sterile air-through a sparger at the rate of '-2 volumes of free 'air' per volume of broth per-minute. Whilethe broth is agitated at a-speed depending uponthetype of agitator, com.- plete sterility'is maintained at all times and the temperature of the brothis-maintained between 24 C. and 30 C. u

The -inventionv may bemore readily understood by a consideration ofwthe pfollowing' illustrative examples;

EXAMPLE F Medium:

G. Wheat gluten. l0 Glycerol l.. l. l0 Distillers solublesflnei as .Sodium chloriden i 5- Tap water to .l.000;ml.;. pH. adjusted-.110;

7 .0 with. .KOH .theniadd- Calcium carbonate l ruuuunn.-- 1

The medium is distr' ute d 50d ml. to? 2.8 liter Fernbachflask, sterilize for 30 minutes-at 121* G. Aftercooling the'm'edi'um is inoculated a suspension" of S'treptemycm netropsis; The

flasks are shaken on a: rotary shaker-shaving a displacement fof =2 '-f;" at 200 R. P. M., at 27 0., fore: days; .At that time, it-wasfound that the broth had a potency of 320 CDU/ml. sThirteen liters of a'mixture of brothand mycehum-was adjusted to pH 2.0. with I-I3PO4, a small amount of Super-eel (a commercial filter aid) was: added and .the mixture filtered. The clear filtrate which had been separated from the mycelium wasadjustedto pH 6.5 with NaOH' and 39 grams of ammonium oxalate was stirredxin and the precipitate of calcium oxalateremoved by filtration. The broth was adjusted to pH 5.5 with HCl and 26 grams of Orange II was added with stirring, whichwas continued for one hour. The dye precipitate was filtered off with the aid of Super-eel, and washed with'2 liters of distilled water. .The air-dried cake was suspended in 2 liters of a.-mixtu reof methanol and 30% acetone and'decomposed bystirring for 1% hours after the addition of 200 ml.of a 50% methanolic solution .;of triethylamine sulfate. ,After filtra tion-, the cake containing Super-cel and the antibioticsulfate was. washed with sufiicient acetone and methanol to remove all traces of dye and excess triethylamine sulfate. The cake was extracted with cold, distilled water and the antibioticsulfate. solution filtered free from the Super-eel. The resulting solution of Netropsin sulfate was dried and found to have a potency of 485 Streptomycin units/mg. .and 745 CDU/mg.

EXAMPLE II Medium: c

Wheat gluten g 10 Glycer01 l g 10 Distillers solubles g 5 3 1 Sodium chloride g 5 Tap water to 1 liter; adjust to pH 7.0 with and add calciumcarbonateu gl Soybean oil ml 1 sterilize minutes at l2lf C. I

Twenty gallons of the above medium in -gallon stirred inoculum tanks were inoculated from a slantof Streptomyoes netropsis and grown for 56 hours at 2'7; C with constant agitation at 1800 R. P. M. while sterile air was blown through the broth at about one volume of free air pervolume of broth per minute. At the end of 56 hours the inoculum prepared as above; was transferred under completely sterile conditions to 150 gallons ofthesamemedium in a 250-gallon fermenter; Agitationandaeration were the same as in the inoculum tank and at theend of 48 hours at 27 C; the broth was found to have a final potency of 320 CDU/ml.

1 One hundred and fifty gallons of broth so prepared was adjusted .to pH 2.0 with sulfuric acid; Super-eel was added and the broth separated from the mycelium by filtration. The clear broth was brought to pH 5.5 with KOH and calcium oxalate, removed by treatment with ammonium oxalate. The calcium oxalate was removed by filtration andOrange II was added-at the rate of 2 grams per liter and stirred for one hour. The dye precipitate was filtered off with the aid of Super-celand washed with'30 g'allons of distilled water.- The dye cake was suspended in 20 gallons of a mixture of 80% acetone and 20% methanoland 250 -ml. of triethylamine sulfate was added. After stirring for 1 hours the-Netropsin sulfate and Super-eel were separated by filtration and the cake washed with 5 gallons of theove mi ed zsu ren a T :c ke was .51

pendedyin- 15 liters of water and grams-of BaClz.2H2O was added to convert the Netropsin. sulfate to the hydrochloride. The solution-of Netropsin hydrochloride was filtered free from Super-eel and barium sulfate, adjusted to pH 2.0 with H2804 which caused the precipitation of excess barium ion as barium sulfate. Excess sulfate ion was removed by stirring with IR-4 (an ion exchange resin sold by the Resinous Products Company) until the pH was 5.5. The Netropsin hydrochloride solution was filtered free from the barium sulfate and the IR-4 resin and dried. The amorphous Netropsin hydrochloride showed a potency of 380 Streptomycin units/mg. and 640 CDU/mg. The amorphous Netropsin hydrochloride was dissolved in distilled water to saturation and on standing crystalline Netropsin hydrochloride separated as fine needles. Recrystallizationwas readily accomplished from water. The crystalline Netropsin hydrochloride assayed at 550 Streptomycin units/mg. and 1200 CDU/- In the foregoing examples it is understood that the compositions of a culture media are merely illustrative and can be varied as, for example, by substituting cottonseed meal or soybean meal for wheat gluten, etc.

Likewise the .conditions of fermentation such as agitation, aeration rate, temperature, etc; can be varied in many ways at once suggested to one skilled in the art. Many alternative methods and variations of the described methods of recovering the antibiotic such as, for instance, solvent extraction at various pHs, adsorption on activated alumina or charcoal and elution with acid or neutral solvents will likewise be considered within the scope of this invention.

Modifications may be made in carrying out this present invention without departing from the spirit and scope thereof and the invention is only to be limited by the appended claims.

We claim;

1. A process for producing Netropsin, which comprises growing Streptomyces netro'psz's in an aqueous nutrient culture medium under submerged aerobic conditions until a substantial antibiotic activity is imparted thereto, separating the mycelium therefrom, and recovering the so-produced Netropsin, from the clear broth.

2. A process for producing Netropsin, which comprises growing a strain of Streptomyces netropsisin an aqueous nutrient culture medium and maintaining the said culture medium under submerged aerobic growth conditions at a term perature of about 25 C. to about 30 C. for a period of severaldays to a week under pure culture conditions, separating the mycelium therefrom, and recovering the so-produced antibiotic, Netropsin, from the clear broth.

3. A process for producing a Netropsin fermentation broth, which comprises cultivating a strain of Streptomyces netropsis in an aqueous nutrient culture medium under aerobic conditions, until substantial antibacterial activity imparted to said solution.

4.- A process as claimed in claim 1, wherein the recovery of the Netropsin includes the'step' of precipitating the antibiotic 'by the addition of an arylazo sulfonic acid dyestufi to the clear broth.

. 5. A substance effective in the group consisting of a base capable of forming salts with acids, whose anhydrous hydrochloride is hygroscopic, shows nooptical activityin inhibiting thegrowth of Gram-negative bacteria, selected from water, methanol or dimethylformamide solutions, has the following elementary analysis:

Per cent 0 42.9 H 5.78

N 28.1 C1 (ionic) 13.7 Cl (total) 13.7

and in a mineral oil suspension exhibits characteristic absorption in the infrared region of the spectrum at the following frequencies expressed in reciprocal centimeters: 3525, 3800, 3170, 1703, 1670, 1583, 1518, 1300, 1244, 1211, 1149, 1104, 1068,

778, 751, 734, 679, and whose hydrated sulfate is sparingly soluble in water and substantially insoluble in the common organic solvents, and in a mineral oil suspension. exhibits characteristic absorption in the infrared. region of the spectrum at approximately the following wave lengths: 3290, 1660, 1630, 1580, 1400, 1285, 1260, 1210, 1145, 1100, 1050, 990, 960, 890, 850, 830 and 805 cmr and the acid salts of said base. 1

6. A hydrochloride of the base defined in claim 5.

7. The base defined in claim 5. 8. A sulfate of the base defined in claim 5.

ALEXANDER C. FINLAY. BEN A. SOBIN.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,422,230 Foster et a1 June 17, 1947 2,449,866 Waksman et al. Sept. 21, 1948 2,461,922 Rake et a1 Feb. 15, 1949 2,462,175 Folkers Feb. 22, 1949 2,482,055 Duggar Sept. 13, 1949 OTHER REFERENCES 

5. A SUBSTANCE EFFECTIVE IN INHIBITING THE GROWTH OF GRAM-NEGATIVE BACTERIA, SELECTED FROM THE GROUP CONSISTING OF A BASE CAPABLE OF FORMING SALTS WITH ACIDS, WHOSE ANHYDROUS HYDROCHLORIDE IS HYGROSCOPIC, SHOWS NO OPTICAL ACTIVITY IN WATER, METHANOL OR DIMETHYLFORMAMIDE SOLUTIONS, HAS THE FOLLOWING ELEMENTARY ANALYSIS: 